The main objective of this application is to provide the PI with research experience and skills which will enable him to function as an independent investigator in the field of cell-based immunotherapy development at the interface between rheumatology and ocular immunology. To this end, the candidate has assembled a mentoring team which includes internationally-recognized leaders in the fields of regulatory T cell (Treg) biology (Juan Lafaille, NYU), rheumatology (Steven Abramson, NYU), and ocular immunology (Rachel Caspi, NIH-NEI). The overarching career goal of the applicant is to develop into a highly qualified physician-scientist with strong basic science skills AND the capacity for rapid translation of immunology concepts to aspects of clinical practice, especially as pertains to the development of new cell-based immunotherapeutic modalities. The proposed project is tailored to these goals through the incorporation and step-wise extension of expertise already acquired by the applicant in clinical and experimental aspects of Behcet's eye disease, and cellular immunology, to new applications, methods and concepts, i.e., experimentation with humanized animal models of inflammatory disease for in vivo proof-of-principle and mechanistic studies. In particular, the project investigates the possibility of massive ex vivo expansion and subsequent adoptive transfer (reinjection therapy) of human regulatory T cell clones for the control of pathogenic effector T cell (Teff) responses in autoimmune uveitis. It tests the main hypothesis that human Treg of the CD4+/CD25hi/CD127lo/Foxp3+/Helios+ phenotype [thymic, or natural (n)Treg] maintain functional stability in vitro and in vivo following massive monoclonal ex vivo expansion, and effectively control general and/or target (uveitis) antigen-related effector T cell responses. The applicant has shown that cloning and massive monoclonal expansion of nTreg under maintenance of in vitro suppressor function is possible, and he now proposes to solidify this approach in Specific Aim 1 and contrast its efficacy with polyclonal, existing approaches. Specific Aim 2 tests the hypothesis that nTreg can be cloned with knowledge of their antigen-specificity. He will use the skills acquired in his previous mentored work in dendritic cell-mediated T cell priming and expansion and incorporate uveitis antigen-specific aspects of immunodominance and pathogenicity as the main educational and scientific objective. Specific Aim 3 focuses on in vivo validation and testing of monoclonally expanded Treg in a 2-tailed approach: Subaim a) will test general immunosuppressive function of clones with unselected antigen-specificity the PI has already generated, in a humanized mouse model of graft-versus-host-disease (GVHD) to determine and quantify in vivo suppression, and subaim b) will establish a humanized experimental autoimmune uveitis (EAU) mouse model for preclinical testing of nTreg clones with uveitis antigen specificity. The knowledge to be gained from the proposed studies bears the potential to significantly improve current approaches of Treg expansion and adoptive transfer strategies, which at present rely mostly on polyclonal techniques and are limited by the unwanted co-expansion of potentially harmful Teff, unstable suppressor function and low expansion rates. The antigen-specific cloning approach further bears the potential of targeted suppression of Teff responses at the site of pathology without significant systemic immunosuppression and eliminates the risks of insertional mutagenesis and dual T cell receptor (TCR) expression inherent in the currently prevailing strategies that confer antigen specificity through lentiviral TCR transduction into polyclonally expanded Treg. Apart from the potential of a new modality of targeted treatment for autoimmune uveitis, results of this research are likely to have a high degree of generalizability to other forms of autoimmune diseases.